ABSTRACT D-alanine-D-alanine is an essential precursor for biosynthesis of peptidoglycan and bacterial cell wall. This dipeptide is synthesized by D-Ala-D-Ala ligase, an enzyme usually encoded by one or two ddl gene(s). Mutants lacking ddl genes are not viable in the absence of D-Ala-D-Ala; it is assumed that such mutants cannot grow in animals due to D-Ala-D-Ala limitation. DdlR, an unusual transcriptional regulator containing an aminotransferase domain, is a positive regulator of the ddl gene in Clostridioides (formerly Clostridium) difficile, an important pathogen that can cause severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. A C. difficile ddlR null mutant retains some activity of D-Ala-D-Ala ligase, but this residual activity is not sufficient for growth in the absence of the dipeptide. We hypothesize that C. difficile strains that lack DdlR will be unable to grow in animals. We propose to explore the potential of DdlR as a novel target for antibacterial therapy and to illuminate prospective drug design by identifying molecules that interact with DdlR and affect its activity. DdlR may be especially well suited as a potential drug target due to its essentiality and expected ability to interact with two different types of small molecules, pyridoxal 5?-phosphate and D-Ala-D-Ala. Moreover, simultaneous targeting of DdlR and D- Ala-D-Ala ligase is a viable possibility. To achieve our goal, we will study in detail the mechanism of DdlR- mediated regulation of D-Ala-D-Ala synthesis in C. difficile using genetic and molecular biological approaches and test the role of DdlR and D-Ala-D-Ala synthesis in cell growth and virulence using a mouse model. Though a very important pathogen itself, C. difficile will also serve as a model for other DdlR-containing pathogenic Gram-positive bacteria, including Bacillus anthracis, Clostridium tetani, Clostridium botulinum, and Clostridium perfringens.